Cyclosporin A inhibits the early phase of NF-kappaB/RelA activation induced by CD28 costimulatory signaling to reduce the IL-2 expression in human peripheral T cells

Cyclosporin A (CsA) inhibits interleukin (IL)-2 production, activation and proliferation of human peripheral T cells (HPTC) costimulated with simultaneous engagement of T cell receptor (TCR)/CD3 and CD28. We demonstrated that 10 ng/ml CsA, which reduced the proliferation of HPTC costimulated with anti-CD3 and anti-CD28 by half, prevented NF-AT and NF-kappaB from migrating into the nucleus. Whereas CsA added even 30 min after the costimulation caused NF-AT to remain in the cytoplasm, the delayed addition of CsA could not prevent NF-kappaB from translocating into the nucleus. CsA, which was added to HPTC simultaneously with the engagement of both CD3 and CD28 or the 1-h-delayed engagement of CD28 after prior TCR/CD3-triggering, inhibited NF-kappaB p65/RelA from binding to the target DNA fragment, followed by reduction of HPTC proliferation in response to the costimulation. When CsA was added 30 min after the delayed engagement of CD28 following the prior engagement of TCR/CD3, these inhibitory effects were diminished. Antisense NF-kappaB p65/RelA oligonucleotides inhibited p65/RelA mRNA expression, diminished IL-2 mRNA expression in the costimulated HPTC and reduced HPTC proliferation to the same extent as CsA added simultaneously with the costimulation. The CsA- or antisense p65/RelA oligonucleotide-induced reduction in the proliferation of costimulated HPTC was overcome by the addition of exogenous IL-2. These findings indicate that the major effects of CsA on the early phase of CD28-mediated costimulation in the presence of TCR/CD3 signaling are to inhibit NF-kappaB/RelA from translocating into the nucleus and binding to the target DNA sequence in the IL-2 gene promoter region, which induces IL-2 expression leading to HPTC proliferation.     

HIV gp41融合多肽对抗原特异性Treg活化的抑制作用

目的探讨HIV gp41融合多肽(fusion peptide,FP)与机体免疫细胞的相互作用,观察FP能否影响抗原特异性的调节性T细胞活化。方法用免疫磁珠分离DO11.10小鼠脾脏CD4+CD25+调节性T细胞(Treg)、CD4+CD25-效应性T细胞(Teff)共培养,3d后采用CCK-8法分析FP对OVA323-339抗原激活的Treg抑制Teff细胞增殖的影响。另外,ELISA法检测FP对Treg细胞IL-10分泌的影响,荧光共聚焦分析FP多肽在Treg细胞表面与TCR的共分布。结果25 mg·L-1的FP能降低Treg的抑制活性和IL-10分泌,在活化的Treg细胞表面有FP和TCR荧光的重叠。结论 FP降低抗原特异性Treg细胞的抑制功能,可能与其抑制IL-10合成有关;它与TCR在细胞膜的相互作用也可能影响APC向Treg细胞递呈活化信号,干扰Treg的活化。     

CD28 co-stimulation regulates the effect of transforming growth factor-beta1 on the proliferation of naïve CD4+ T cells

Transforming growth factor-beta1 (TGF-beta1) is a critical regulator of T cell responses in vivo. In vitro, TGF-beta1 can either enhance or inhibit T cell proliferative responses, but the relevant factors that determine the T cell response to TGF-beta1 remain obscure. Here, we present evidence that CD28 co-stimulation modifies the effects of TGF-beta1 on T cell proliferation. In the absence of CD28 co-stimulation, TGF-beta1 potently suppressed TCR-stimulated proliferation of na?ve T cells. In the presence of CD28 co-stimulation, TGF-beta1 potently inhibited T cell apoptosis and enhanced TCR-stimulated proliferation. A similar effect of CD28 co-stimulation was not observed in memory/effector cells, whose proliferation was enhanced by TGF-beta1, whether co-stimulated or not. We examined the mechanism by which CD28 modulates na?ve T cell responses to TGF-beta1. Since CD28 co-stimulation classically is a potent enhancer of interleukin (IL)-2 production, we anticipated observing high IL-2 production from na?ve T cells stimulated with anti-CD3/anti-CD28 and TGF-beta1. Surprisingly, however, TGF-beta1 strongly inhibited production of IL-2 from na?ve CD4(+) T cells, even when CD28 was engaged. Even though IL-2 levels were strongly suppressed by TGF-beta1 to trace levels, antibody neutralization studies showed that IL-2 is still a basic requirement for the proliferation of anti-CD3/anti-CD28/TGF-beta1-stimulated na?ve T cells. These data show that CD28's modulation of T cell responses to TGF-beta1 is not via the production of high levels of IL-2, and suggest that engagement of CD28 may activate additional downstream pathways that modulate the responses of na?ve T cells to TGF-beta1.     

Astragaloside II triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity

Aim:

To investigate the immunomodulating activity of astragalosides, the active compounds from a traditional tonic herb Astragalus membranaceus Bge, and to explore the molecular mechanisms underlying the actions, focusing on CD45 protein tyrosine phosphatase (CD45 PTPase), which plays a critical role in T lymphocyte activation.

Methods:

Primary splenocytes and T cells were prepared from mice. CD45 PTPase activity was assessed using a colorimetric assay. Cell proliferation was measured using a [³H]-thymidine incorporation assay. Cytokine proteins and mRNAs were examined with ELISA and RT-PCR, respectively. Activation markers, including CD25 and CD69, were analyzed using flow cytometry. Activation of LCK (Tyr505) was detected using Western blot analysis. Mice were injected with the immunosuppressant cyclophosphamide (CTX, 80 mg/kg), and administered astragaloside II (50 mg/kg).

Results:

Astragaloside I, II, III, and IV concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the EC₅₀ values ranged from 3.33 to 10.42 μg/mL. Astragaloside II (10 and 30 nmol/L) significantly enhanced the proliferation of primary splenocytes induced by ConA, alloantigen or anti-CD3. Astragaloside II (30 nmol/L) significantly increased IL-2 and IFN-γ secretion, upregulated the mRNA levels of IFN-γ and T-bet in primary splenocytes, and promoted CD25 and CD69 expression on primary CD4⁺ T cells upon TCR stimulation. Furthermore, astragaloside II (100 nmol/L) promoted CD45-mediated dephosphorylation of LCK (Tyr505) in primary T cells, which could be blocked by a specific CD45 PTPase inhibitor. In CTX-induced immunosuppressed mice, oral administration of astragaloside II restored the proliferation of splenic T cells and the production of IFN-γ and IL-2. However, astragaloside II had no apparent effects on B cell proliferation.

Conclusion:

Astragaloside II enhances T cell activation by regulating the activity of CD45 PTPase, which may explain why Astragalus membranaceus Bge is used as a tonic herb in treating immunosuppressive diseases.     

Suppressive effect of a novel water-soluble artemisinin derivative SM905 on T cell activation and proliferation in vitro and in vivo

Artemisinin and its derivatives exhibit potent immunosuppressive activity. The aim of this study was to investigate the suppressive effects of SM905, a new water-soluble artemisinin derivative, on T lymphocytes both in vitro and in vivo, and explore its potential mode of action. The results showed that SM905 had a high inhibitory activity in Concanavalin A (ConA)-induced splenocyte proliferation and mixed lymphocyte reaction, and a relatively low cytotoxicity in vitro. In ovalbumin-immunized mice, oral administration of SM905 dose-dependently suppressed T cell proliferative response to ovalbumin, and inhibited anti-ovalbumin interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production by T cells. Further studies showed that SM905 inhibited TCR (T cell receptor)/CD3 plus CD28-mediated primary T cell proliferation and cytokine production (IL-2 and IFN-gamma), and exerted an inhibitory action on the phosphorylation of mitogen-activated protein (MAP) kinases including extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal kinase (JNK), and the activation of Ras. The results of this study provided experimental evidence that the new artemisinin derivative SM905 had immunosuppressive effects both in vitro and in vivo. SM905 suppressed T cell activation, which was associated with the inhibition of MAP kinases and Ras activation. Our results suggested a potential of SM905 to be developed as a new type agent for treating T cell-mediated immune disorder.     

Influence of Endogenous Opioid Systems on T Lymphocytes as Assessed by the Knockout of Mu, Delta and Kappa Opioid Receptors

Here, we evaluated the influence of endogenous opioid activation on immune responses by examining consequences of all three opioid receptor gene (mu, delta and kappa) inactivation. In triple-opioid receptor knockout mice, splenocytes and thymocytes numbers, lymphocyte subsets as well as proliferation and cytokines induced by in vitro stimulation of T lymphocytes were measured. Compared with wild-type mice, similar lymphocyte distribution in thymus and spleen as well as comparable T lymphocyte proliferation were observed, while lower levels of IL-2 and IFNγ as well as higher levels of IL-4 and IL-10 were found in triple-opioid receptor knockout mice. Together, our results indicate a shift from TH1 to TH2 cytokines in triple-opioid receptor knockout animals, suggesting that global endogenous opioid tone drives T lymphocytes toward a TH1 profile under non-pathological conditions.     

咖啡酸苯乙酯对小鼠CD3~+T细胞体外活化和增殖的影响

目的研究咖啡酸苯乙酯(caffeic acid phenethyl ester,CAPE)对小鼠CD3+T淋巴细胞活化及增殖的影响,探讨其作用机制。方法无菌分离小鼠淋巴结淋巴细胞,制备淋巴细胞悬液。在淋巴细胞活化实验中,分别与不同浓度的咖啡酸苯乙酯预先孵育2h后,加入促分裂原伴刀豆蛋白A(ConA),运用荧光抗体染色技术结合流式细胞术,8h后,检测T淋巴细胞早期活化标记分子CD69的表达情况;24h后,检测T淋巴细胞中期活化标记分子CD25的表达情况;在淋巴细胞增殖试验中,运用CFDA-SE标记法检测T淋巴细胞刺激48h后的增殖情况。结果CAPE(0·5、1、5、10mg·L-1)能够明显地抑制ConA刺激的小鼠淋巴细胞活化,且呈浓度依赖性;CAPE(0·5、1、5、10mg·L-1)能够明显地抑制ConA刺激的小鼠淋巴细胞增殖,且呈浓度依赖性。结论CAPE对小鼠CD3+T淋巴细胞的体外活化和增殖具有抑制作用,其作用机制可能通过同时抑制PLC-γ信号途径和MAP激酶途径,由此推理CAPE是一种潜在的有效的免疫抑制剂。     

黄芪多糖对烧伤小鼠细胞免疫功能的作用

应用小鼠烧伤模型，对黄芪多糖（ＡＰＳ）的免疫增强作用进行了体内外研究。结果表明：体内应用ＡＰＳ（２５０ｍｇ·ｋｇ－１，ｑｄ，连续５ｄ），可明显提高烧伤小鼠Ｔ淋巴细胞转化，ＩＬ－２的产生及ＩＬ－２Ｒ的表达；体外分别应用５０、１００、２５０ｍｇ·Ｌ－１的黄芪多糖，发现其可纠正烧伤小鼠Ｔ淋巴细胞转化，ＩＬ－２的产生及ＩＬ－２Ｒ表达的受抑状态，并促进巨噬细胞产生ＩＬ－１，抑制ＰＧＥ２合成，且呈剂量依赖关系；体外去除烧伤小鼠脾细胞中的巨噬细胞后，ＡＰＳ对Ｔ淋巴细胞转化，ＩＬ－２产生及ＩＬ－２Ｒ表达的调节作用消失。提示ＡＰＳ对烧伤小鼠的免疫调节作用依赖于巨噬细胞，通过调节其分泌ＩＬ－１，抑制ＰＧＥ２合成，而促进ＩＬ－２产生及ＩＬ－２Ｒ表达，进而增强Ｔ淋巴细胞增殖。     

Lumin,a cyanine dye,enhances interleukin 12-dependent interferon gamma production by lipopolysaccharide-stimulated mouse splenocytes

Lumin was orally administered to mice daily for 3 d, and on the day following the final administration, mice were sacrificed and splenocytes were stimulated with lipopolysaccharide (LPS). Splenocytes obtained from lumin-treated mice showed enhanced production of interferon gamma (IFN-gamma) and increased percentages of CD3+ cells. Although T cells are considered to be the source of IFN-gamma, it is unlikely that LPS directly stimulates T cells. Next we performed neutralization experiments using a monoclonal antibody (mAb) against interleukin (IL-)12 because this cytokine, which is produced by macrophages, has the direct ability to induce IFN-gamma production and the proliferation of activated T cells. This antibody inhibited IFN-gamma production by splenocytes. We thus show that orally administered lumin enhances IFN-gamma production by splenocytes when the latter are stimulated with LPS, a phenomenon that was observed in correlation with activation of T cells by IL-12, that is produced by macrophages.     

Trichloroethylene enhances TCR-CD3-induced proliferation of CD8(+) rather than CD4(+) T cells

Trichloroethylene (TCE) is suspected as a potent immunomodulator that accelerates the development of allergic diseases. We previously reported that TCE promotes ovalbumin (OVA)-induced active cutaneous anaphylaxis, including enhancing antigen-specific serum IgE levels and splenic lymphocyte proliferation. However, the target cells and molecular mechanism through which TCE modulates antigen-specific immune responses remain unclear. To identify a potential underlying mechanism, we investigated whether TCE modulates T cell receptor (TCR)-induced T cell activation and proliferation in vitro. TCE enhanced T cell proliferation primed by anti-CD3 antibody, but not concanavalin A, in a dose-dependent fashion. In addition, TCE enhanced anti-CD3-primed proliferation of CD8(+) rather than CD4(+) T cells. Consistent with this result, TCE markedly enhanced the Lck phosphorylation mediated by anti-CD3 antibody in CD8(+) but not CD4(+) T cells. Furthermore, we analyzed the effect of TCE exposure via drinking water for 2 weeks on splenocyte populations in non-immunized and OVA-immunized mice. In OVA-immunized mice, TCE (3 mg/l) significantly expanded CD3(+), CD8(+) and CD4(+) cell populations, however the effect at the lower concentration was significant only in the CD8(+) populations, whereas TCE had no effect on these cells population in non-immunized mice. These findings suggest that TCE enhances TCR-CD3-induced proliferation of CD8(+) rather than CD4(+) cells and disrupts various activities of peripheral T cells.     

Rottlerin inhibits human T cell responses

Rottlerin is a pharmacological inhibitor of protein kinase C (PKC) theta, a novel PKC selectively expressed in T lymphocytes. PKC theta is known to regulate T cell receptor (TCR)/CD28 signalling pathways in T lymphocytes, but the impact of PKC theta inhibition on human T cell responses remains undefined. In this work, we describe the effects of rottlerin on the responses of CD4+ and CD8+ human T lymphocytes upon polyclonal activation. We observed a dose-dependent inhibition of CD4+ and CD8+ T cell proliferation in response to anti-CD3/anti-CD28 antibodies stimulation in the presence of rottlerin. This inhibition was associated with impaired CD25 expression and decreased interleukin (IL)-2 production in activated T cells. In contrast, rottlerin did not alter IL-2-induced T cell proliferation. Furthermore, we demonstrated that rottlerin blocked interferon (IFN) gamma, IL-10 and IL-13 mRNA expression in TCR/CD28 activated CD4+ T cells. These findings place rottlerin as a potent immunosuppressive agent for the development of novel therapies in T cell mediated immune disorders.     

Hypothemycin inhibits the proliferative response and modulates the production of cytokines during T cell activation

Hypothemycin, a resorcylic acid lactone antibiotic, was identified as active in a screen for inhibitors of T cell activation. It was found to inhibit the proliferation of mouse and human T cells stimulated with anti-CD3 mAb + PMA and of human PBMC stimulated with anti-CD3 mAb alone. This inhibition was partially reversed by exogenous IL-2 indicating that it is not due to non-specific toxicity. Hypothemycin potently suppressed the production of IL-2 (IC50: 9 nM) but affected IL-2-induced proliferation to a lesser extent (IC50: 194 nM). Hypothemycin also inhibited IL-6, IL-10, IFN-gamma and TNF-alpha production. By contrast, it markedly enhanced the production of IL-4, IL-5 and IL-13. These effects were seen both at the mRNA and protein secretion levels. Analysis of the effect of hypothemycin on CD69 induction suggested that it disrupts calcineurin-independent rather than calcineurin-dependent signaling. Furthermore, hypothemycin was able to inhibit the phosphorylation of ERK1/2 induced by PMA treatment of T cells. Therefore, hypothemycin represents an inhibitor of T cell activation with a novel mode of action and unique modulatory activity on cytokine production.     

Inhibition of lymphocyte activation and function by the prenylation inhibitor L-778,123

Prenylated Ras GTPases transduce signals from the T cell receptor, CD28 costimulatory receptor and IL-2 receptor. Since signals from these receptors mediate T cell activation, proliferation and survival, we hypothesized that the prenylation inhibitor L-778,123 would impart immunomodulation.The effect of L-778,123 on T cell activation (CD71 or CD25 surface expression) was determined by flow cytometry. Peripheral blood mononuclear cell (PBMC) proliferation in the presence of L-778,123 and/or cyclosporine (CsA) was determined by [3H]thymidine incorporation. The ability of L-778,123 to inhibit IL-2 receptor signaling was investigated by measuring IL-2 induced proliferation in CTLL-2 cells and IL-2 prevention of apoptosis in activated human PBMC. L-778,123 inhibited lectin induced expression of CD71 and CD25 with IC50's of 6.48 +/- 1.31 microM and 84.1 +/- 50.0 microM, respectively. PBMC proliferation was inhibited by L-778,123 with an IC50 of 0.92 +/- 0.23 microM, and addition of CsA did not increase the potency. L-778,123 did not inhibit IL-2 and IFN-gamma production by T cells. L-778,123 abrogated IL-2 induced proliferation of CTLL-2 cells with an IC50 of 0.81 +/- 0.44 microM. However, L-778,123 minimally reversed the prosurvival effect of IL-2 in activated lymphocytes. IL-2 ligand and receptor production during T cell activation are relatively unaffected by L-778,123. However, the activation and proliferative effects of IL-2 on T cells are potently blocked by L-778,123. These results reveal a selective blockade of the IL-2 cytokine axis distal to the IL-2 receptor by the L-778,123 and warrant evaluation of prenylation inhibitors in treating transplant rejection and autoimmune diseases.     

CD8 blockade promotes the expansion of antigen-specific CD4+ FOXP3+ regulatory T cells in vivo

Treatment with a cocktail of CD4 and CD8-specific monoclonal antibodies (mAb) induces long-term transplantation tolerance and regulatory CD4(+) T cells that induce tolerance in non-tolerant T cells. In contrast, treatment with a CD4-specific mAb alone fails to induce long-term tolerance. The current study was designed to test the hypothesis that CD8 blockade plays a role in promoting the development of CD4(+) regulatory T cells. Using the DO11.10 CD4(+) TCR transgenic mouse model we show that treatment with a CD4/CD8-specific mAb cocktail induces antigen-specific tolerance to OVA, measured by a significant decrease in OVA-specific IgG, on challenge with antigen. Although treatment with OVA and the CD4-specific mAb alone also induces a significant decrease in OVA-specific antibody, the number of DO11.10 cells is significantly greater in mice treated with the CD4/CD8-specific mAb cocktail, and this is associated with a significant increase in proliferation of DO11.10 cells in response to specific antigen. DO11.10 cells sorted from mice made tolerant to OVA with the CD4/CD8-specific mAb cocktail promote an OVA-specific IgG1 (Th2-type) response but not an OVA-specific IgG3 (Th1-type) response on transfer into new syngeneic recipients, suggesting their ability to regulate the type of antigen-specific immune response that ensues after priming with antigen. In addition, DO11.10 cells from tolerant mice express markers that are characteristic of CD4(+) regulatory cells, including FOXP3, GITR and CTLA4, but not CD25. Taken as a whole, these data suggest that CD8 blockade promotes CD4(+) FOXP3(+) regulatory CD4(+) T cells by promoting their proliferation in tolerant mice.     

Ginsenoside Rg1 enhances CD4(+) T-cell activities and modulates Th1/Th2 differentiation

Panax ginseng is commonly used as a tonic medicine in Asian countries such as Korea, China, and Japan. It has been reported that ginsenoside Rg1 in P. ginseng increases the proportion of T helper (Th) cells among the total number of T cells and promotes IL-2 gene expression in murine splenocytes. This implies that ginsenoside Rg1 increases the immune activity of CD4(+) T cells, however, the exact mechanism remains unknown. The present study elucidated the direct effect of Rg1 on helper T-cell activities and on Th1/Th2 lineage development. The results demonstrated that ginsenoside Rg1 had no mitogenic effects on unstimulated CD4(+) T cells, but augmented CD4(+) T-cell proliferation upon activation with anti-CD3/anti-CD28 antibodies in a dose-dependent manner. Rg1 also enhanced the expression of cell surface protein CD69 on CD4(+) T cells. In Th0 condition, ginsenoside Rg1 increases the expression of IL-2 mRNA, and enhances the expression of IL-4 mRNA on CD4(+) T cells, suggesting that Rg1 prefers to induce Th2 lineage development. In addition, ginsenoside Rg1 increases IL-4 secretion in CD4(+) T cells under Th2 skewed condition, while decreasing IFN-gamma secretion of cells in Th1 polarizing condition. Thus, Rg1 enhances Th2 lineage development from the na?ve CD4(+) T cell both by increasing Th2 specific cytokine secretion and by repressing Th1 specific cytokine production. Therefore, these results suggest that ginsenoside Rg1 is a desirable agent for enhancing CD4(+) T-cell activity, as well as the correction of Th1-dominant pathological disorders.     

Involvement of protein kinase A in histamine-mediated inhibition of IL-2 mRNA expression in mouse splenocytes

The release of histamine from mast cells and basophils during allergic reactions can regulate functions of T cells and may influence the nature of the immune response to a given antigen. The effects of histamine on T lymphocytes are associated with its binding to H2-receptors linked with adenylate cyclase, elevation of cAMP levels and activation of cAMP-dependent protein kinase (PKA). In this report we explore the role of PKA in histamine-mediated effects on IL-2 mRNA expression and IL-2 protein secretion. Fresh isolated mouse splenocytes (C57Bl/6) were pretreated with histamine (10(-4) M) for 1 h in the presence or absence of Rp-cAMPS (50 microM), an inhibitor of PKA regulatory subunit. The cells were then washed thoroughly and activated with plate-bound anti-CD3 (5 microg/ml), or PHA (1:100) or PMA + ionomycin (10 ng/ml, 1 microg/ml) for 6 h. Pretreatment with histamine inhibited IL-2 mRNA expression and secretion in cells activated with anti-CD3 or PMA, but not in cells activated with PMA + ionomycin. Rp-cAMPS prevented histamine-mediated suppression and did not itself affect IL-2 production. These results provide evidence that histamine affected IL-2 production when the cells were activated via the T cell receptor (TCR)/CD3 complex, but did not interfere with signal transduction pathways downstream of PKC leading to production of IL-2. These effects of histamine on IL-2 secretion and mRNA expression were mediated via PKA.     

Patterns of A2A extracellular adenosine receptor expression in different functional subsets of human peripheral T cells. Flow cytometry studies with anti-A2A receptor monoclonal antibodies

Signaling through A2A adenosine receptors (A2AR) regulates T lymphocyte expansion and modulates T cell receptor (TCR)-mediated effector functions in vitro. To understand the role of A2ARs in the regulation of immune response, we investigated the expression levels of this receptor in different functional lymphocyte subsets. Monoclonal anti-A2AR antibody was used to develop a flow cytometric assay to quantify the expression A2ARs on lymphocytes. We report that detectable levels of expression of A2ARs are much higher among T cells than B cells. More CD4(+) than CD8(+) T cells express A2ARs, but activation of T cells increases A2AR expression, predominantly in CD8(+) T cells. No significant differences were found in the proportion of A2AR+ cells between CD8(low) and CD8(high) T cells or between TCR/CD3(low) and TCR/CD3(high) T cells. Studies of T helper cell subsets (TH1 and TH2) reveal that lymphokine-producing cells are much more likely to express A2ARs than are cells that do not produce lymphokines. These results suggest that A2ARs are variably expressed on T cell subsets and may regulate cytokine production in activated T lymphocytes.     

Immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the<Emphasis Type="Italic">In Vitro</Emphasis> immune response in pristane-induced lupus mice

Systemic lupus erythematosus (SLE) is characterized by overactive B cells that differentiate into autoantibody-forming cells, aberrant T cell function that provides helping B cells produce autoantibodies, and overproduction of proinflammatory cytokines. However, immunodysregulation in lupus pathogenensis remains incomplete. We examined mitogen-stimulated production of proinflammatory cytokines, cell proliferation, T cell activation, and T cell apoptosis in vitro in pristane-induced lupus BALB/c mice compared to normal mice. LPS-stimulated production of IL-6 and IL-10 by splenocytes and macrophages from pristane-induced lupus mice were remarkably up-regulated compared to normal mice, whereas production of macrophage TNF-alpha was significantly down-regulated. Moreover, in vitro production of IL-2, IL-6, IL-10 and IFN-gamma by Con A-stimulated splenocytes, cell proliferation in LPS- or Con A-stimulated- thymocytes and splenocytes, and expression of CD69+CD4+ T cells in Con A-stimulated splenocytes were greatly increased in cells derived from pristane-induced lupus mice compared to normal mice. In addition, splenic T cells and CD4+ T cells in thymocytes from pristane-induced lupus mice were more resistant than nonautoimmune normal cells to Con A-induced apoptosis. Our findings indicate that immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the in vitro immune responses in pristane-induced lupus mice may explain some of lupus pathogenesis.